New blood vessel development in the ovary, uterus and placenta, which is critical to ultimate pregnancy outcome, is regulated by estradiol-17p (E2) acting through estrogen receptor a (ERa). The underlying processes include direct actions of E2 on endothelial cells to promote their growth and migration. These mechanisms also mediate cardiovascular protection by E2 remote from pregnancy. However, the basis for direct regulation of endothelial cell phenotype by E2 and ERa is poorly understood. We have shown that E2 stimulates endothelial NO synthase (eNOS) via the activation of pjasma membrane-associated ERa, and that these receptors are G protein-coupled. In preliminary studies with a new estrogen-dendrimer conjugate (EDC), we have observed that G-protein-mediated membrane ERa activation of Src and eNOS underlies E2- induced endothelial cell growth and migration. The OBJECTIVE of the proposed research is to determine if estrogen regulates endothelial cell phenotype via direct coupling of membrane ERato G proteins. In recent initial experiments we have identified dynamic protein-protein interactions between recombinant ERa and Gai and G(3y. Aim 1 is to determine the molecular basis of ERa-G protein interaction and Src activation. The domains of ERa mediating G protein interactions will be determined, and their roles in signaling to Src will be tested. The ability of peptide sequences derived from the ERa interaction domains to inhibit signaling to Src will also be assessed. Aim 2 is to determine if membrane ERa coupling to G proteins regulates endothelial cell growth and migration in vitro. Using EDC as a membrane-directed ligand, the requirement for ERa will be determined by gain- and loss-of-function strategies, and additional interventions will test the requirements for Gai and Gpy activation. Involvement of ERa-Gai and ERa-Gpy interactions will be tested using interfering peptides identified in Aim 1. Aim 3 is to determine if membrane ERa coupling to G proteins regulates endothelial cell migration in vivo. In a nonpregnant female mouse model of carotid artery reendothelialization, responses to E2 versus EDC will be evaluated in wild-type versus ERa"''mice. The requirement for Ga and Gpy activation will be tested by local blockade of their functions in the carotid artery, and the requirement for ER-Gai and ER-Gpy interactions will be assessed by endothelial delivery of interfering peptides. Aim 4 is to identify nuclear targets of membrane ERa-G protein activation in endothelium in vivo. Using focused microarrays and RT-PCR, changes in gene expression will be determined in endothelium sorted from Tie2-GFP;ERa+/+versus Tie2-GFP;ERa'/"mice treated with E2 versus EDC. Genes downstream of G protein activation will be identified by pertussis toxin treatment, and the requirement for ER-Gai and ER-Gpy interactions will be tested by endothelial delivery of interfering peptides. The roles of identified target genes will be tested in gain- and loss-of-function studies of cultured endothelial cell phenotypes. By investigating the novel, most proximal processes by which E2 and ERa regulate endothelial cell phenotype, the proposed research will increase our fundamental understanding of how vascular actions of estrogen promote the reproductive and nonreproductive health of women.